This invention relates to an animal model that exhibits polyglutamine toxicity, and more particularly to methods for identifying genes that modulate polyglutamine toxicity using Drosophila.
Expansion of polyCAG tracts is associated with human hereditary neurodegenerative disorders and neuronal toxicity (Kaytor et al., J Bio. Chem., 274:37507-37510 (1999)). Huntington""s disease and several other hereditary neurodegenerative disorders are characterized by expansion of a polyglutamine sequence (LaSpada et al., Nature, 352:77-79 (1991); Koide et al., Nat. Genet., 6:9-13 (1994); Kawaguchi et al., Nat. Genet., 8:221-228 (1994); Orr et al., Nat. Genet., 4:221-226 (1993); Sanpei et al., Nat. Genet., 14:277-284 (1996); and Zhuchenko et al., Nat. Genet., 15:62-69 (1997)). The expanded polyCAG tracts encode abnormally long polyglutamine sequences within specific proteins promoting their nuclear and/or cytoplasmic aggregation. The protein aggregation is believed to contribute to cellular toxicity including cell death or apoptosis (Trottier et al., Nature, 378:403-406 (1995); Davies et al., Cell, 90:537-548 (1997); and DiFiglia et al., Science, 277:1990-1993 (1997)).
The mechanism of toxicity and cell death by expanded polyglutamines is not yet fully understood. Peptides containing expanded polyglutamine tracts are prone to forming cytoplasmic (CIs) and/or nuclear inclusions (NIs). Two variables appear as major determinants of the aggregation propensity, subcellular localization or toxicity of polyglutamine-containing peptides. The relative length of the polyglutamine tract determines the aggregation propensity and cytotoxicity; the longer it is, the more likely it is to form inclusions and cause cell death. The overall size of the peptide determines subcellular localization as well as aggregation propensity and cytotoxicity; shorter, truncated gene products with expanded repeats are more likely to form inclusions, and these inclusions are more likely to be in the nucleus than in the cytoplasm. These inclusions occasionally recruit their full-length counterpart.
Perinuclear inclusions produced by truncated huntingtin peptides recruit endogenous huntingtin in transfected human kidney epithelial 293Tcells (HEK 293T). Cotransfection of truncated ataxin-3 (SCA3 gene product) with its full-length counterpart, containing either a normal or an expanded polyglutamine tract, resulted in the recruitment of either of the two full-length proteins into perinuclear inclusions formed by the truncated ataxin-3. However, this type of recruitment was not observed in HD brains. In another set of experiments, huntingtin was recruited to neuritic plaques, neurofibrillary tangles and dystrophic neurites in Alzheimer""s disease, and to Pick bodies found in Pick disease. Heteromerous aggregates were also formed between co-expressed ataxin-1, with normal or expanded polyglutamine, and ataxin-3 with an expanded polyglutamine repeat in transfected HEK 293T.
Experiments in mouse striatal cell culture and transgenic mice suggested that nuclear localization was necessary for the pathogenic effects. On the other hand, experiments in a human embryonic kidney cell line suggested that polyglutamine can be equally cytotoxic in the cytoplasm or the nucleus. Furthermore, in cultured mouse clonal striatal cells or in SCA1 transgenic mice, aggregation of polyglutamines appeared to be neither sufficient nor necessary for pathogenesis. When NI formation was suppressed in neurons transfected with mutant huntingtin, cell death increased.
The molecular components of the pathways involved in neuronal degeneration and protein aggregation have been investigated. These include: components of protein folding (Cummings et al., Nat Genet, 19:148-154 (1998); Wyttenbach et al., Proc. Natl. Acad Sci. USA, 97:2898-2903 (2000); and Kobayashi et al., J. Biol. Chem., 275:8772-8778 (2000)), protein degradation (Chai et al., Hum. Mol. Genet., 8:673-682 (1999)), gene expression (Boutell et al., Hum. Mol. Genet., 8:1647-1655 (1999); Kazantsev et at, Proc. Natl. Acad. Sci. USA, 96:11404-11409 (1999); and Li et al., J. Neurosci., 19:5159-5172 (1999)), and programmed cell death (Portera et al., J. Neurosci., 3775-3787 (1995); Wellington et al., J Biol. Chem., 273:9158-9167 (1998); and Ona et al., Nature, 399:263-267 (1999)), as well as interacting proteins (Kaichman et al., Nat, Genet, 16:44-53 (1997); Sittler. et al., Mol. Cell, 2:427-436 (1998); Waragai et al., Hum. Mol Genet., 8:977-987 (1999)), neurotransmitters, and their receptors (Cha et al., Proc. Natl. Acad. Sci. USA, 95:6480-6485 (1998); Chen et a., J. Neurosci., 72:1890-1898 (1999); and Reynolds et al., J. Neurochem., 72:1773-1776 (1999)). A Drosophila model has recapitulated abnormal protein aggregation and neuronal toxicity associated with polyglutamine disorders, and a candidate heat shock gene has been shown to have a suppressing effect (Warrick et a., Cell, 93:939-949 (1998); Jackson et al., Neuron, 21:633-642 (1998); Marsh et al., Hum. Mol Genet., 9:13-25 (2000); and Kazemi-Esfarjani, Science, 287:1837-1840 (2000)). The present invention is based upon an alternative animal model that mimics polyglutamine and/or protein folding abnormalities observed in humans.
The present invention relates to an animal model useful for identifying molecules that modulate expression or activity of proteins involved in polyglutamine toxicity, neuronal and other degenerative disorders, cancer and other proliferative disorders in humans. This animal model is also useful for identifying molecules that modulate disorders associated with undesirable or aberrant protein folding, aggregation, degradation or aberrant transport. Such molecules include genes and other compounds that modulate protein aggregation or folding and associated disorders, including polyglutamine toxicity and polyglutamine related disorders.
A genetic screen using a Drosophila animal model of the invention identified in vivo genetic modulators of polyglutamine toxicity. Three Drosophila genes, heat shock protein 40/HDJ1 (dHDJ1), tetratricopeptide repeat protein 2 (dTPR2) and myeloid leukemia factor 1 (dMLF), were capable of decreasing polyglutamine toxicity in affected flies. Thus, the Drosophila genes or their mammalian homologues and other compounds identified using an in vivo animal model of the invention can be used as therapeutics in treating polyglutamine toxicity and associated disorders in humans. A method of the invention, and the genes and compounds identified, are also applicable for the identification and treatment of disorders associated with other diseases that result from or are associated with intracellular or extracellular protein misfolding/aggregation. Particular examples include Alzheimer""s disease, Parkinson""s disease, Creutzfeldt-Jacob""s disease (CJD), bovine spongiform encephalopathy, Huntington""s disease (HD), Machado-Joseph disease (MJD), Spinocerebellar ataxias (SCA), dentatorubropallidoluysian atophy (DRPLA), Kennedy""s disease, stroke and head trauma. In addition, as the human homologues of dTPR2 and DMLF (TPR2 and MLF, respectively) are associated with tumorigenesis (neurofibromatosis 1) and leukemias (myelodysplastic syndrome and acute myeloid leukemias), respectively, these genes, and the flies carrying dTPR2 and dMLF P-element insertions or their transgenic versions, will be helpful in identifying cancer therapeutics.
In accordance with the present invention, there are provided methods of screening for genes or compounds that modulate polyglutamine toxicity. In one embodiment, a method of the invention includes providing a first animal expressing a polyglutamine sequence, wherein the sequence produces polyglutamine toxicity in the animal; breeding the first animal to a second animal, wherein the second animal has a marker sequence inserted into its germline, thereby producing progeny; screening the progeny for increased or decreased polyglutamine toxicity relative to the first animal thereby identifying a progeny having increased or decreased polyglutamine toxicity; and identifying one or more genes adjacent to or having an insertion of the marker sequence that confers increased or decreased polyglutamine toxicity in the progeny having increased or decreased polyglutamine toxicity. In another embodiment, a method further includes identifying a mammalian homologue (e.g., human homologue) of the gene.
Methods of screening that are included employ first and second animal invertebrates. In one embodiment, a method includes invertebrates of the genus Drosophila (e.g., Drosophila melanogaster).
In one embodiment, a marker used in the methods and animals of the invention includes a P element sequence. In another embodiment, the marker sequence comprises a polynucleotide sequence that disrupts or alters expression of one or more genes near the sequence. In yet another embodiment, a marker sequence includes an expression control element conferring expression of the one or more genes near the marker. In one aspect, the expression control element increases expression. In another aspect, the expression control element decreases expression.
Methods of the invention include screening methods in which a plurality of second animals having markers located at different positions within their genome are screened. Thus, in one embodiment, a second animal is selected from a group of two or more animals having markers inserted into different locations of its genomic DNA. In another embodiment, the second animal is selected from a group of 10 to 100, 100 to 500, or 500 or more of the animals. In yet another embodiment, the second animal is selected from a library of animals having markers inserted at random locations of their genomic DNA. In still another embodiment, each of the second animals is generated by random P-element insertions into the genome. In one aspect, a library of animals is generated by random P element insertion.
Polyglutamine sequences of the methods and transgenic animals of the invention include, for example, sequences having between about 35 to 50 glutamine residues, between about 50 to 100 glutamine residues, between about 100 to 150 glutamine residues and having about 150 or more glutamine residues. The sequences can be encoded by a plurality of CAGs, CAAs or a combination thereof. Expression of the plurality of CAGs, CAAs or combination thereof can be conferred by a constitutive, regulatable or tissue specific expression control element. In one embodiment, the regulatable element comprises an inducible or repressible element. In another embodiment, the regulatable element comprises a GAL4 responsive sequence. In yet another embodiment, the tissue specific element confers neural, retinal, muscle or mesoderm cell expression.
Polyglutamine sequences can additionally include other molecular entities. In one embodiment, a polyglutamine sequence further includes a tag. In one aspect, a tag comprises an epitope tag. In another aspect, a tag comprises a hemagglutinin sequence.
Animals of the invention include progeny animals produced by the screening methods of the invention that employ animals. In one embodiment, a progeny animal exhibits decreased polyglutamine toxicity relative to a parent that exhibits polyglutamine toxicity. In another embodiment, a progeny animal exhibits increased polyglutamine toxicity relative to a parent that exhibits polyglutamine toxicity.
Animals of the invention further include transgenic animals including a transgene containing a plurality of CAGs and at least one CAA sequence encoding a polyglutamine repeat sequence. In one embodiment, a transgenic animal is an invertebrate. In another embodiment, a transgenic animal is of the genus Drosophila (e.g., Drosophila melanogaster).
Transgenic animals of the invention including a transgene containing a plurality of CAGs and at least one CAA sequence encoding a polyglutamine repeat sequence can have any number of CAGs and CAAs in any ratio encoding the repeat sequence. In one embodiment, the number of GAGs to GAAs is in ratio of between about 1:1 and 2:1. In another embodiment, the number of GAGs to GAAs is in ratio of between about 2:1 and 5:1. In yet another embodiment, the number of GAGs to GAAs is in ratio of between about 5:1 and 10:1. In still another embodiment, the number of GAGs to GAAs is in ratio of between about 10:1 and 50:1.
Thus, a transgenic animal of the invention including a transgene containing a plurality of CAGs and at least one CAA sequence encoding a polyglutamine repeat sequence can express a polyglutamine repeat sequence of any length. In one embodiment, the polyglutamine sequence is between about 5 and 20 amino acids in length. In another embodiment, the polyglutamine sequence is between about 20 and 50 amino acids in length. In yet another embodiment, the polyglutamine sequence is between about 50 and 100 amino acids in length. In additional embodiments, the polyglutamine sequence is between about 100 and 200 amino acids in length, between about 100 and 500 amino acids in length and between about 50 and 200 amino acids in length. In various aspects, a polyglutamine sequence further includes a tag (e.g., epitope, hemagluttinin, etc.).
In other embodiments, expression of the polyglutamine sequence in the transgenic animals of the invention is conferred by a constitutive, regulatable or tissue specific expression control element. In one aspect, a tissue specific expression control element confers neural, retinal, muscle or mesoderm cell expression. In another aspect, a tissue specific expression control element comprises an Appl or rhodopsin 1 promoter or GLASS transcription factor element.
Transgenic animals of the invention further include animals having a polyglutamine sequence of sufficient length to produce toxicity in one or more cells, tissue or organs of the animal. In one embodiment, toxicity is produced in a neuron cell or brain. In another embodiment, toxicity is produced in a retinal cell or eye. In additional embodiments, toxicity is produced in muscle and mesoderm. Such animals can further include a gene that increases or decreases polyglutamine toxicity produced in the cell, tissue or organ. In one embodiment, such an animal includes a marker sequence inserted into its genomic DNA, wherein the marker is located adjacent to a gene or inserted into a gene whose expression or activity increases or decreases polyglutamine toxicity in the animal. In one aspect, the marker sequence is near or inserted into a gene containing a J domain. In another aspect, the marker sequence is near or inserted into HDJ1. In yet another aspect, the marker sequence is near or inserted into TPR2. In still another aspect, the marker sequence is near or inserted into MLF gene.
Thus, methods for identifying a compound or transactivating factor that modulates polyglutamine toxicity in an animal also are provided. In one embodiment, a method includes contacting an animal that exhibits polyglutamine toxicity with a test compound; and determining whether the test compound increases or decreases polyglutamine toxicity in the animal. Increased or decreased polyglutamine toxicity identifies the test compound as a compound that modulates polyglutamine toxicity. The compound may be present in the animal""s food or drink or administered to a tissue or organ of the animal (directly or indirectly).
In addition, methods of producing a transgenic animal characterized by polyglutamine toxicity are provided. In one embodiment, a method includes transforming an animal embryo or egg with a transgene comprising a plurality of CAA and CAG sequences encoding a polyglutamine sequence having sufficient length to produce polyglutamine toxicity in the animal produced from the embryo or egg; and selecting an animal that exhibits polyglutamine toxicity in one or more cells or tissues. Polyglutamine sequences need only be of a length (or sequence where other non-glutamine residues are present) to produce toxicity in one or more cells, tissue or organs of the animal. Animal produced by these methods include transgenic animals of the invention.
Compositions including isolated polynucleotides and polypeptides are also provided. In one embodiment, a polypeptide or a polynucleotide encodes a polypeptide that decreases polyglutamine toxicity. In one embodiment, a polynucleotide sequence has about 65% or more identity to a Drosophila TPR2 (dTPR2) sequence set forth as SEQ. ID NO:2, with the proviso that the sequence is distinct from the EST sequences set forth in FIG. 11. In another embodiment, a polynucleotide sequence has about 65% or more identity to a Drosophila MLF (dMLF) sequence set forth as SEQ. ID NO:4, with the proviso that the sequence is distinct from the EST sequences set forth in FIG. 12. Functional subsequences of TPR2 and MLF that decrease polyglutamine toxicity also are provided.
Invention polynucleotides can be operatively linked to an expression control element. In one embodiment, an expression control element confers expression in a cell, organ or tissue that has or is at risk of having polyglutamine toxicity. In one aspect, an expression control element confers expression in neuron, eye, muscle or mesoderm. In additional aspects, an expression control element is an Appl or rhodopsin 1 promoter or GLASS transcription factor element.
Further provided are isolated polynucleotide sequences that to invention Drosophila TPR2 (dTPR2) set forth as SEQ. ID NO:2, and dMLF set forth as SEQ. ID NO:4, sequences. In one embodiment, a sequence hybridizes to a Drosophila TPR2 (dTPR2) sequence set forth as SEQ. ID NO:2 under moderately stringent or highly stringent conditions, with the proviso that the sequence is distinct from the EST sequences set forth in FIG. 11. In another embodiment, a sequence hybridizes to a Drosophila MLF (dMLF) set forth as SEQ. ID NO:4 under moderately stringent or highly stringent conditions, with the proviso that the sequence is distinct from the EST sequences set forth in FIG. 12.
Such polynucleotide sequences can be of any length, and include, inter alia, polynucleotide having 20 or more contiguous nucleotides, polynucleotide having 30 or more contiguous nucleotides, polynucleotide having 40 or more contiguous nucleotides, polynucleotide having 50 or more contiguous nucleotides, etc.
Such sequences further include sequences that encode polypeptides, including functional polypeptides as described herein. In one embodiment, a sequence encodes a subsequence of TPR2 that decreases polyglutamine toxicity. In another embodiment, a sequence encodes a subsequence of MLF that decreases polyglutamine toxicity. Expression of such sequences can be conferred by an expression control element, for tissue specific expression, for example. Polypeptides encoded by such sequences also are provided.
Compositions of the invention further include mammalian (e.g., human) homologues of the genes that modulate polyglutamine toxicity in an animal as described herein operatively linked to an expression control element in a pharmaceutically acceptable carrier. In one embodiment, a composition includes a polynucleotide sequence encoding a human MLF polypeptide operatively linked to an expression control element in a pharmaceutically acceptable carrier. In another embodiment, a composition includes a polynucleotide sequence encoding a human TPR2 polypeptide operatively linked to an expression control element in a pharmaceutically acceptable carrier. In additional embodiments, expression control elements confer expression of the mammalian (e.g., human) homologue in a cell, tissue or organ of a subject, having or at risk of having polyglutamine toxicity or a polyglutamine related disorder, as described herein.
Methods of identifying compounds or trans-activating protein factors that modulate expression or activity of a target dHDJ1, dTPR and dMLF also are provided. In one embodiment, a target gene is screened by transforming host cells with a promoter or regulatory region of the target gene operatively linked to a reporter construct. In various aspects, a promoter or regulatory region of the target gene includes a sequence set forth in any of SEQ ID NO:s:9, 10 or 11. Candidate target gene promoters and regulatory regions also include promoter or regulatory regions of mammalian (e.g., human) homologues of dHDJ1, dTPR2 and dMLF.
In another embodiment, a method includes incubating components containing HDJ1, TPR2 and MLF polypeptide or subsequence thereof, or a cell or animal expressing HDJ1, TPR2 and MLF polypeptide or subsequence thereof, and a test compound, under conditions sufficient to allow the components to interact. The effect of the test compound on HDJ1, TPR2 and MLF polypeptide activity (e.g., polyglutamine toxicity) or expression is then determined.
In yet another embodiment, transactivating factors are identified using the polynucleotides of the invention in vitro or in a cell-based assay. A method includes contacting a promoter or regulatory region of a target gene of HDJ1, TPR2 or MLF (e.g., a sequence set forth in any of SEQ ID NO:s:9, 10 or 11) with a candidate factor and determining whether the factor bins to the promoter or regulatory region. The invention methods therefore include in vitro, cell-based and in vivo methods to screen for effector compounds, transacting factors or binding proteins. Such methods are useful for identifying transactivating factors or other compounds that modulate HDJ1, TPR2 or MLF expression and are therefore applicable in methods of identifying treatments as well as the treatment methods described herein.
Methods of increasing survival of a cell having or at risk of having polyglutamine toxicity are also provided. In one embodiment, a method includes contacting the cell with an amount of TPR2 or MLF polypeptide sequence, or a polynucleotide sequence encoding TPR2 or MLF polypeptide, to increase survival of the cell. Such methods include in vitro, ex vivo and in vivo, and where the cell is a neural, retinal, muscle or mesoderm cell.
Methods of decreasing apoptosis of a cell also are provided. In one embodiment, a method includes contacting the cell with an amount of TPR2 or MLF polypeptide sequence or a polynucleotide sequence encoding TPR2 or MLF polypeptide to decrease apoptosis of the cell. Such methods include in vitro, ex vivo and in vivo, and where the cell is a neural, retinal, muscle or mesoderm cell.
Methods of decreasing polyglutamine toxicity in a cell having or at risk of having also are provided. In one embodiment, a method includes contacting the cell with an amount of J domain containing polypeptide, TPR2 or MLF polypeptide sequence, or a polynucleotide sequence encoding the J domain containing polypeptide, TPR2 or MLF polypeptide sequence to decrease polyglutamine toxicity in the cell. The toxicity may be decreased by decreasing cell death or apoptosis. The toxicity may be decreased by decreasing protein aggregation, increasing transport or folding, etc.
Such in vitro, ex vivo and in vivo methods include where the cell is a neural, retinal, muscle or mesoderm cell. Thus, methods of decreasing polyglutamine toxicity in a tissue or organ of a subject having or at risk polyglutamine toxicity also are provided. In one embodiment, a method includes contacting the cell, tissue or organ with an amount of a J domain containing polypeptide, a TPR2 or MLF polypeptide sequence, or a polynucleotide sequence encoding the J domain containing polypeptide, TPR2 or MLF polypeptide, to decrease polyglutamine toxicity in the cell, tissue or organ of the subject. In various aspects, the tissue is brain, eye, muscle or mesoderm.
Methods of decreasing the severity of a frontotemporal dementia, prion disease, polyglutamine disorder or protein aggregation disorder in a subject having or at risk of a frontotemporal dementia, prion disease, polyglutamine disorder or protein aggregation disorder are provided. In one embodiment, a method includes administering to the subject an amount of J domain containing polypeptide, a TPR2 or MLF polypeptide sequence, or a polynucleotide sequence encoding the J domain containing polypeptide, TPR2 or MLF polypeptide, to decease the severity of the frontotemporal dementia, prion disease, polyglutamine disorder or protein aggregation disorder in the subject.
Methods of treatment include prophylactic administration. Disorders treatable include neurological and muscle disorders and disorders that impair long term or short term memory or coordination of the subject. Disorders treatable also include disorders characterized by the presence of protein aggregates, amyloid plaques, degeneration or atrophy in an affected tissue or organ.
Particular disorders treatable by the methods of the invention include Alzheimer""s disease, Parkinson""s disease, Creutzfeldt-Jacob""s disease (CJD), bovine spongiform encephalopathy, Huntington""s disease (HD), Machado-Joseph disease (MJD), Spinocerebellar ataxias (SCA), dentatorubropallidoluysian atophy (DRPLA), Kennedy""s disease, stroke and head trauma. The severity is decreased by decreasing cell death or apoptosis, increasing cell survival, decreasing protein aggregation, increasing protein folding, transport, etc. Severity is also decreased by slowing the progression or reversing one or more symptoms of the disorder (e.g., decreasing memory loss, improving memory, decreasing loss of coordination, improving coordination).
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.